Effectiveness of Nanoparticle-Based Acidulated Phosphate Fluoride (APF) Gel on Surface Enamel Fluoride Uptake: an Interventional Study

Statement of the Problem: Despite topical fluoride being used for over 50 years in caries prevention, its complete potential in terms of formation of fluorapatite enamel and prolonged surface retention has not been harnessed. Purpose: This study aimed to assess the effectiveness of nanoparticle based acidulated phosphate fluoride (APF) gel on surface enamel fluoride uptake through split mouth design over a period of six months, on patients undergoing orthodontic treatment and indicated for bilateral extraction of premolars on at least one of the arch. Materials and Method: A split mouth non randomized clinical trial was performed. Each of the 30 participants received one application of 4 minutes duration of both the intervention: Right half of the mouth received nanoparticle based APF gel, and left half of the mouth received conventional APF gel (16 Oz Pascal Corp.; strawberry flavor). Bilateral acid etch biopsy of enamel surface was take at 3 intervals- baseline,24 hours and 30 days. Using 1ml of 0.5M perchloric acid, acid etch enamel biopsy was taken for all the study subjects at 3 intervals of time (baseline, 24 hours and 30 days), bilaterally on the buccal and palatal/lingual surface of maxillary or mandibular premolars indicated for extraction, using 1 (l of 0.5M perchloric acid. After the premolars were extracted, scanning electron microscope (SEM) analysis was done to determine the surface characteristics of enamel in both groups. Results: Overall, both fluoride uptake and depth of biopsy remained significant even after controlling for the covariates (time, group) individually and simultaneously (p< 0.05). Further analysis showed that fluoride uptake was significantly increased and biopsy depth significantly decreased in the nanoparticle based APF gel group at 24 hour and 30 day evaluation respectively. Conclusion: By reducing the size of sodium fluoride to increase the surface enamel uptake, our results support the use of this top down approach as a promising strategy for effective delivery of topical fluorides. This highlights that the top down approach of nanoscience to reduce the size active compound sodium fluoride has increased the uptake and retention of surface enamel fluoride.


Introduction
Over 50 decades, fluorides have been the cornerstone in the prevention of dental caries, and have proven benefits on both topical and systemic supplementation [1]. The term 'topically applied fluoride' refers to those delivery systems which provide fluoride to exposed surfaces of the dentition, at elevated concentrations, for a local protective effect and are therefore not intended for ingestion [2]. In the present scenario of evidence based practice, the American Dental Association [3] substantiates the efficacy of the acidulated phosphate fluoride (APF) gel as a one of the cost effective means among the various professionally applied topical fluoride (PATF) agents, next to fluoride varnish [3].
Despite the proven effectiveness of fluoride gels in caries prevention, there still remains a gap between the quantity of fluoride supplemented and the quantity taken up by the enamel surface which is in turn responsible for the prevention of demineralization [4]. In order to overcome this, alternative strategies have been tried to improve the fluoride uptake like altering the temperature of the topical fluoride agent, surface treatment of the enamel with phosphoric acid, prophylaxis of teeth before application, combining chelating metallic ions and laser irradiation of the enamel surface [2].
Although numerous improvements have been made, economic level implications of these when applied on a community wide basis is questionable. Technological advancements such as the use of nanoscience, works on particles of dimension one hundredth of a micrometer [5]. First described by Dr. Richard P Feyman in 1959, nanotechnology has made a paradigm shift in all the areas of dentistry from prevention to restorative and surgical materials [5]. However, in preventive dentistry, it has been largely restricted to the alteration of the oral biofilm by addition of metallic nanoparticles to existing fluoride varnishes, remineralizing agents like casein phosphate protein or restorative materials like dentin bonding agents, composites, and cements [6].
Moreover, many authors have suggested that the improvement in fluoride therapy should be such that the formulation results in increased concentrations of permanently bound fluoride [3,[7][8]. One other possible way, which is still unexplored in improving the enamel uptake of fluoride from gels, is altering the particle size of the active component which is sodium fluoride (NaF). This sodium fluoride compound has an octahedral lattice of 462pm with each particle measuring approximately 99.47µm in diameter. Literature evidence shows that over 90% of this fluoride is freely available as calcium fluoride, which predominantly dissolves with only a small fraction being available in bound form of fluorapatite within the enamel crystals [2].
In vitro and in vivo studies using APF gel have reported an immediate increase in fluoride uptake following topical application, which gradually decreased with increasing depth or time of measurement [7][8]. An indirect method of this surface enamel fluoride uptake was through measuring the surface microhardness of the extracted tooth samples. An average increase of 40-60% in the hardness values, 24 hours following the application of APF gel has been demonstrated from in vitro, in situ and ex vivo models by researchers across the globe in both deciduous and permanent dentitions [9][10]. Our previous study on extracted teeth also showed similar results at 24 hour evaluation, which gradually decreased during the one month observation period [11]. However, the inability of these models to simulate oral conditions implies the need for better in vivo studies to assess the fluoride uptake from topical gels by the enamel surface. Hence, it was hypothesized that the reduced particle size of sodium fluoride and incorporation in freshly prepared APF gel will alter the fluoride uptake compared to the conventional gel, which may in turn affect the fluoride retention on the enamel surface.

Trial description
A bilingual written informed consent was obtained from the participants of age above 18 years and parental consent was obtained for participants of age 15 to 18 years.
Permission to conduct the study was obtained from the  In addition, it was ensured that the participants were permanent residents of Chennai where the optimal fluoride levels in the drinking water is restricted to 1 ppm were. All participants who had systemic disorders and/ or who were under medications, who were indicated for orthodontic correction without extraction of premolar teeth, extraction of any teeth other than premolars or any other method of correction of teeth were excluded.
Other reasons for exclusion were the presence of developmental defects of enamel, caries or restoration present on premolars and those who developed any form of acute dental problems during the study period.

Sample size
The sample size for each group was calculated using the G power statistical software. The mean values from our previous study [11] were taken into consideration for sample size estimation as microhardness is assumed to be proportional to the amount of fluoride uptake.
The power of the study was kept at 80%, with an alpha error of 0.5 and the estimates were calculated to 95% confidence interval. In order to account for loss to follow up, during the course of the study and based on the availability, 30 participants were included in the study. Figure 2 represents the flow of participants through the study.  to be significant.

Results
The mean age of the study population was 22.  Table 3 represents the distribution of mean fluoride        were equivalent in their effectiveness [16]. Time dependent fluoride acquisition from APF gel was tested in laboratory and in vivo conditions [17][18]. The authors concluded that the fluoride concentrations in the enamel was maintained high during the treatment periods and sustained for an average of 2 to 10 months after which the levels stabilized at 1600 ppm [17][18]. They found that fluoride uptake significantly increased with increase in the contact with topical agent and recommended professional application time of 4 minutes.
Few authors concluded that presence of unclean teeth did not affect fluoride uptake or the caries inhibitory properties of fluoride but only the time and labor intensity required per patient was drastically reduced in the absence of prophylaxis procedure [19][20][21]. This was effectively found in our study, where the maximum time taken for APF gel application and the enamel biopsy together was less than 10 minutes for each half of the mouth, with an interval of 30 minutes in between, after which the patient was asked to rinse thoroughly. This ensured that cross over effect of one agent over the other is minimized.
The uptake and retention of fluoride on the enamel surface had been assessed through a variety of methods amongst which many modifications have been tried for the acid biopsy technique predominantly differing in terms of with/ without the use of burs (silicon carbide, felt burs, impregnated burs) or the type and concentration of acid used (hydrochloric, perchloric acid) [7,13,[16][17][18][19][20][21][22][23][24][25][26]. In our present study, the field biopsy technique [13] was used as it did not involve any invasive procedure and was performed only on specific demarcated area and not the whole teeth.
Applications of laser and cold atmospheric plasma have also been tried in the recent past in combination with APF gel have also showed promising results in increasing fluoride uptake [22][23][24]. In line with these advancements, a commendable stride has been made with the introduction of nanoscience in healthcare [6]. A randomized, double blinded in-situ trial found that the combination of nanohydroxyapatite crystals with sodium fluoride (NaF) on dentin remineralization improved fluoride delivery into early dentinal lesions and prevented demineralization [25].
Our present study showed an overall increase in the fluoride uptake in both groups following APF gel application. However, the average increase in the nanoparticle based group was higher than the control at 24 hour evaluation which gradually returned close to baseline at the end of 30 days in both the groups. This was similar to the mean fluoride concentration of 6520±407 ppm, 15 minutes after gel application [16].
On comparing the biopsy depth at different time points, our study showed a minimal but gradual significant decrease from 71.15±3.18µm to 69.33±2.11µm in the nanoparticle based group whereas, similar estimation in the conventional group was not significant.
Clinical estimation of the depth of enamel biopsy has been done in literature earlier [16]. However, the above estimations were done on maxillary incisors of children of 8 to 12 years which is different from our study population.

Similar observations in extracted teeth samples in
in-situ conditions for APF was found to be around 2.3 to 9.1µ in the 1 to 5 layers of enamel over an etching period of 120 seconds [26]. This could also be not considered equivalent as the assessments were done on extracted teeth samples mounted on prosthetic appliances which was worn by 12 volunteers.
In vitro experiment on 528 enamel specimens to assess uptake and retention of APF, thixotropic and pluoronic gel showed an average presence of 6929±6091 ppm, 5913±10,135ppm and 3566±1463ppm of fluoride respectively immediately at 24 hours, 7 and 28 days evaluation with a mean biopsy depth of 3.02±1.19µm for the APF group [27]. A cumulative etching depth of 54µm after an etching period of 20 minutes with 0.1M HCl acid has also been reported [28].

Conclusion
The present study highlights the effective application of nanotechnology in preventive dentistry by modifying the particle size of sodium fluoride compound in APF gel. Further evaluation of this nanoparticle based gel in terms of maximum achievable reduction in particle size, alteration in duration of application, frequency of application and use of chelating compounds should be done to substantiate its long-term effectiveness.